Succession of Microbial Community in a Small Water Body within the Alluvial Aquifer of a Large River

Round 1

Reviewer 1 Report

The described research is interesting and is worthy of publication.  The major item of concern with the article is with Figs. 3, 4, and 6 which have meaningless "Sample" numbers on the x-axis.  There was no mention in the text when each of the samples were collected or the exact location of each.  It is suggested that sample dates (day, month and year) are listed along the x-axis.  It is suspected that some of the samples were collected in different calendar years which should be justified in the methods,  results, and discussion sections since interannual variations are typical.  

Author Response

Firstly, we would like to thank the reviewers and the editor for their invaluable input in improving this MS. Herein, we provide point-by-point answers to all reviewers' remarks and suggestions.

Answers to reviewer #1

Reviewer #1

The described research is interesting and is worthy of publication. The major item of concern with the article is with Figs. 3, 4, and 6 which have meaningless "Sample" numbers on the x-axis. There was no mention in the text when each of the samples were collected or the exact location of each. It is suggested that sample dates (day, month and year) are listed along the x-axis. It is suspected that some of the samples were collected in different calendar years which should be justified in the methods, results, and discussion sections since interannual variations are typical.

The major item of concern with the article is with Figs. 3, 4, and 6 which have meaningless "Sample" numbers on the x-axis. It is suggested that sample dates (day, month and year) are listed along the x-axis. 

The „sample“ numbers on the x-axis on Figs. 3, 4, and 6 have been changed into months and year of sampling according to the reviewer's suggestion.

There was no mention in the text when each of the samples were collected or the exact location of each.

The sampling procedure was corrected according to the reviewer's suggestion as follows:

Line 120. Phytoplankton and bacterial samples were taken monthly from June 2017 until March 2018 on the deepest point of the gravel pit using the vertical Hydro-Bios water sampler (Hydro-Bios Apparatebau GmbH, Germany).

It is suspected that some of the samples were collected in different calendar years which should be justified in the methods, results, and discussion sections since interannual variations are typical.

We fully agree with the reviewer that the interannual variatons play an important part of every investigation. This was corrected by placing months and year of sampling in the graphical representations of the results as well as in the methodology part of the MS. To clarify, the sampling started on late spring (June) of 2017, as this was also the beginning of the project, and lasted throughout summer, autumn, winter and early spring of 2018, in order to collect the samples from each season and to obtain a representative dataset, for the same reason the reviewer also pointed out. The results were presented in the MS according to the sampling dynamics, as was also the discussion.

Reviewer 2 Report

The authors have made an exhaustive study in a small water body. Although the influence of nitrogen on primary production is well documented the value of the present study lies on the information provided for this particular water body in order to serve as a base for future monitoring.

It is worth publishing in its present form

Author Response

Decision: MAJOR REVISION

Firstly, we would like to thank the reviewers and the editor for their invaluable input in improving this MS. Herein, we provide point-by-point answers to all reviewers' remarks and suggestions.

Reviewer #2

The authors have made an exhaustive study in a small water body. Although the influence of nitrogen on primary production is well documented the value of the present study lies on the information provided for this particular water body in order to serve as a base for future monitoring. It is worth publishing in its present form.

We thank the reviewer for his comments.

Reviewer 3 Report

In this paper, the authors intend to investigate the potential of small water bodies in the utilization of nitrogen compounds in the Drava River alluvial area. The topic is interesting and data on the role of small water bodies in alluvial lowlands are still scarce. However, what the authors are claiming in the title is not supported by the results presented. The contribution of the gravel pit in the utilization of nitrates cannot be observed from data in Table 1, in which only the Min and Max numbers during the investigated period of 10 months are showed. And the changes in the concentrations of environmental variables may be normal fluctuations as environmental factors (temperature, pH) change during a year. Moreover, only one point is selected as the study area and only one sample site is set in this area, which lacks representation obviously. In the introduction part, the background is too long and is not very connected to the focus of this study. And figures in the article should be optimized. In figure 1-7, what does S01-S012 mean? Therefore, I think the manuscript is not well prepared and the publication in this journal is not recommended.

Author Response

Firstly, we would like to thank the reviewers and the editor for their invaluable input in improving this MS. Herein, we provide point-by-point answers to all reviewers' remarks and suggestions.

Reviewer #3

In this paper, the authors intend to investigate the potential of small water bodies in the utilization of nitrogen compounds in the Drava River alluvial area. The topic is interesting and data on the role of small water bodies in alluvial lowlands are still scarce. However, what the authors are claiming in the title is not supported by the results presented. The contribution of the gravel pit in the utilization of nitrates cannot be observed from data in Table 1, in which only the Min and Max numbers during the investigated period of 10 months are showed. And the changes in the concentrations of environmental variables may be normal fluctuations as environmental factors (temperature, pH) change during a year. Moreover, only one point is selected as the study area and only one sample site is set in this area, which lacks representation obviously. In the introduction part, the background is too long and is not very connected to the focus of this study. And figures in the article should be optimized. In figure 1-7, what does S01-S012 mean? Therefore, I think the manuscript is not well prepared and the publication in this journal is not recommended.

However, what the authors are claiming in the title is not supported by the results presented.

We agree with this fact, therefore we have suggested a modification to the title as follows:

„Importance of small water body in the recycling of nitrogen in the alluvial area of a large river“

The contribution of the gravel pit in the utilization of nitrates cannot be observed from data in Table 1, in which only the Min and Max numbers during the investigated period of 10 months are showed. And the changes in the concentrations of environmental variables may be normal fluctuations as environmental factors (temperature, pH) change during a year.

We acknowledge this remark made by the reviewer, and have made changes in the manuscript accordingly, i.e. we have completely removed or, in some cases, modified parts of MS where the terms of utilization, modulation of nitrogen or other nutrients by the microbial commnity were mentioned, since these was not the focus of our study, nor were they corroborated by the results. The modifications to the text are as follows:

Title: „Importance of small water body in the recycling of nitrogen in the alluvial area of a large river“

Introduction:

Lines 91-95. To investigate the role of these small water bodies in the nitrogen recycling in the Drava River alluvial area, we have selected a small inactive gravel pit. By employing interdisciplinary approaches, we aim to characterize ecosystem functionality, emphasizing bacterial and phytoplankton diversity, and their effects on nitrogen recycling along the hydrological transport pathways.

Discussion:

Lines 433-435. Since nitrogen fertilizers are widely used in agriculture to increase crop production, the cropping practices and soil texture have been found to influence the extent of nitrate leaching [74].

Conclusion:

Lines 578-579. Nitrogen compounds likely control phytoplankton biomass, thereby influencing the complete microbial community structure.

Moreover, only one point is selected as the study area and only one sample site is set in this area, which lacks representation obviously.

We thank the reviewer for this comment. As both the focus (the influence of nitrogen on microbial community of a small water body in the alluvial area of Drava River) and the workflow (10 month sampling) were set by default in the project, we were obliged to follow this without making big adjustments or changes. However, we strongly agree with the reviewer about the necessity to increase both temporal and spatial scale of this study in future investigations.

In the introduction part, the background is too long and is not very connected to the focus of this study.

We agree with the reviewer's comments. Several parts of the introduction have been completely removed and were not enlisted in here. Several parts were also rewritten and modified to make a stronger focus to the presented study. The modifications, which are enlisted into the revised version of the MS, are as follows:

Lines 41-45. Various aspects of nutrient dynamics in freshwater ecosystems are of paramount importance for understanding how the productivity of surface waters is controlled and provide the opportunity to analyse current and future impacts of anthropogenic activities on freshwater ecosystems. In such environments, a large part of primary production may depend on the recycling of nutrients such as nitrogen compounds [1]. Nitrogen is an essential element that often limits growth in aquatic ecosystems, and a key compound in many biochemical processes that are important for life, but can be harmful in high concentrations [2-4].

Lines 60-62. The composition of the microbial community depends on environmental conditions that may affect ecosystem function [16-19], as they drive the various processes of recycling, dynamics and assimilation of nitrogen compounds in freshwater habitats [18-20].

And figures in the article should be optimized. In figure 1-7, what does S01-S012 mean?

Follwoing the reviewer's comment, we have made changes to optimize the figures. The „sample“ numbers (S01-S012) on all pertaining figures (2, 3A, 3B, 4, 5, 6, and 7) have been changed into months and year of sampling.

Round 2

Reviewer 1 Report

The revisions made by authors greatly improved the manuscript.  One interesting aspect not covered in the study would be sampling some local water wells to verify the chemical content without the plankton.

Author Response

Reviewer #1

The revisions made by authors greatly improved the manuscript.  One interesting aspect not covered in the study would be sampling some local water wells to verify the chemical content without the plankton.

Answer:

We are thankful to the reviewer for his comments. We agree with his remark, indeed one part of the project, under which this manuscript was done, also covers sampling of several piezometers and private water wells where stable water isotopes were measured in order to set up a conceptual model of the aquifer. The data acquired will greatly help in understanding groundwater flow and geochemical processes within the Drava River aquifer.

Reviewer 3 Report

After considering the changes the authors have made in the manuscript, publication in its current form is recommended.

Author Response

Reviewer #3

After considering the changes the authors have made in the manuscript, publication in its current form is recommended.

Answer:

We are thankful to the reviewer for his comments.